A sulforhodamine peroxynitrite probe containing an indole base ring, and a preparation method and application thereof

Abstract

The application belongs to the field of fluorescent sensing, and discloses a sulfur rhodamine peroxynitrite probe containing an indole bottom ring, a preparation method and application thereof. The new compound SR-In of the application is not fluorescent due to PET effect, and generates a new fluorescent substance, thioxanthone, after reacting with peroxynitrite. Since the spectral range of sulfur rhodamine and thioxanthone is completely different, the fluorescent signal can be greatly changed before and after detection, and high-sensitivity detection of peroxynitrite is realized. In addition, the indole bottom ring in the structure of SR-In can only be combined with peroxynitrite and oxidized into thioxanthone, so SR-In does not react with other common active oxygen in the organism, and good peroxynitrite selectivity is shown.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence sensing, specifically relating to a thirodane peroxynitrite probe containing an indole base ring, its preparation method, and its application. Background Technology

[0002] peroxynitrite (OONO) - Nitrite is a reactive oxygen species produced by the coupling reaction of nitric oxide and superoxide radicals in vivo. Studies have shown that nitrite can react with biomolecules such as proteins and nucleic acids, thereby affecting intracellular physiological activities and even leading to cell necrosis or apoptosis. For example, nitrite can alter cell signal transduction pathways through nitration, thereby regulating cellular physiological processes. Furthermore, diseases such as Alzheimer's disease, cancer, circulatory shock, and reperfusion injury are also associated with nitrite imbalance. Therefore, developing in-situ detection methods for nitrite is crucial for understanding its function in complex biological systems. However, the short half-life (<10 ms), low concentration, and high reactivity of nitrite make its detection under physiological conditions a significant challenge. Although various methods for nitrite detection have been reported, fluorescence imaging techniques using fluorescent probes have attracted increasing attention due to their simplicity, high sensitivity, high selectivity, and in-situ detection capabilities.

[0003] The James research group at the University of Bath, UK, reported a peroxynitrite probe based on arylboronic esters, which exhibited a 6.5-fold fluorescence enhancement upon reaction with peroxynitrite (Chem. Commun., 2017, 53, 12822-12825). The Yoon Joo-young research group at Ewha Womans University, South Korea, reported a peroxynitrite probe based on p-aminophenol, which exhibited a 14-fold fluorescence enhancement upon reaction with peroxynitrite (Anal. Chem., 2017, 89, 16, 8496-8500). However, these probes suffer from insufficient fluorescence signal response and low selectivity for peroxynitrite. Therefore, developing fluorescent peroxynitrite probes with novel detection mechanisms is of great significance. Summary of the Invention

[0004] The main objective of this invention is to overcome the shortcomings of existing technologies and provide a method for constructing thiorhodamine containing an indole base ring, which can be applied to the detection of peroxynitrite.

[0005] The technical solution of this invention:

[0006] A thirodane peroxynitrite probe SR-In containing an indole base ring has the following structure:

[0007]

[0008] A method for preparing a thirodane peroxynitrite probe containing an indole base ring, comprising the following steps:

[0009] 1) Mix 3,3'-thiobis(N,N-dimethylaniline) with indole-3-carboxaldehyde and dissolve in an acidic solvent;

[0010] 2) Stir the solution obtained in step 1) under high temperature conditions, then pour it into ice water to neutralize the acidic reaction solution with sodium carbonate;

[0011] 3) After extraction with an organic solvent, the organic phase is dried and the organic solvent is removed by vacuum distillation to obtain the crude product;

[0012] 4) The crude product was separated and purified by silica gel column chromatography with eluent to obtain the thiodamine peroxynitrite probe SR-In containing an indole base ring.

[0013] The synthesis route is as follows:

[0014]

[0015] In step 1), the molar ratio of 3,3'-thiobis(N,N-dimethylaniline) to 3-indolecarboxaldehyde is 1:1-5.

[0016] In step 1), the acidic solvent is one of methanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, propionic acid, concentrated sulfuric acid, or trifluoroacetic acid, and the concentration of 3,3'-thiobis(N,N-dimethylaniline) in the acidic solvent is 20-100 mg / mL.

[0017] In step 2), the reaction temperature is 100-140℃ and the reaction time is 2-8 hours.

[0018] In step 3), the organic solvent used for extraction is one of dichloromethane, chloroform, or ethyl acetate.

[0019] In step 4), the eluent is methanol and dichloromethane in a volume ratio of 1:15-30.

[0020] An application of a thirodamine peroxynitrite probe SR-In containing an indole underring ring in the detection of peroxynitrite: In the absence of peroxynitrite, the thirodamine peroxynitrite probe SR-In exhibits no fluorescence when excited at 405 nm; however, in the presence of peroxynitrite, the probe molecule is oxidized to thioxanthrone by the peroxynitrite, at which point the probe exhibits strong fluorescence in the 450-600 nm range when excited at 405 nm, thus enabling the detection of peroxynitrite.

[0021] The beneficial effects of this invention are as follows: The novel compound SR-In itself is non-fluorescent due to the PET effect. However, upon reaction with peroxynitrite, it generates a new fluorescent substance—thioxanthone. Since thiorhodamine and thioxanthone have completely different spectral ranges, the fluorescence signal can be significantly altered before and after detection, achieving highly sensitive detection of peroxynitrite. Furthermore, the indole base ring in the SR-In structure can only be bound to and oxidized to thioxanthone by peroxynitrite. Therefore, SR-In does not react with other common reactive oxygen species in organisms (superoxide anion, hydrogen peroxide, hydroxyl radicals, hypochlorite), exhibiting excellent peroxynitrite selectivity. Attached Figure Description

[0022] Figure 1 The image shows the 1H NMR spectrum of the target compound SR-In obtained in Example 1.

[0023] Figure 2 This is a high-resolution mass spectrum of the target compound SR-In obtained in Example 1.

[0024] Figure 3 This is the UV-Vis absorption spectrum of SR-In before and after the addition of peroxynitrite as described in Example 4.

[0025] Figure 4 This is the fluorescence emission spectrum of SR-In before and after the addition of peroxynitrite as described in Example 4.

[0026] Figure 5 This is a bar chart of the fluorescence intensity of SR-In after the addition of different interfering substances in Example 5.

[0027] Figure 6 This is a schematic diagram showing the excellent selectivity of SR-In for peroxynitrite in Example 5. Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0029] Example 1: Synthesis of thirodane peroxynitrite probe SR-In containing an indole base ring

[0030] 3,3'-Thiobis(N,N-dimethylaniline) (54.5 mg, 0.2 mmol), 3-indolecarboxaldehyde (87.1 mg, 0.6 mmol), and 2 mL of formic acid were added sequentially to a thick-walled, pressure-resistant tube, and the reaction was carried out at 120 °C for 2 hours. After the reaction was completed, the reaction solution was poured into 20 mL of ice water and neutralized with sodium carbonate. Dichloromethane (3 × 20 mL) was added for extraction, and the solution was dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using eluent (methanol:dichloromethane = 1:25) to obtain SR-In (31.2 mg, 37%).

[0031] The product was characterized by proton nuclear magnetic resonance spectroscopy. 1 1H NMR (400MHz, CDCl3) δ 13.11 (s, 1H), 7.89 (d, J = 9.7Hz, 2H), 7.81 (d, J = 8.2Hz, 1H), 7.60 (d, J = 2.7Hz, 1H), 7.20 (t, J = 7.5Hz, 1H), 7.04 (dt, J = 14.8, 7.8Hz, 2H), 6.95 (d, J = 2.5Hz, 2H), 6.68 (dd, J = 9.8, 2.5Hz, 2H), 3.21 (s, 12H). Among these, the proton peak with a chemical shift of 3.21 ppm is the proton peak of N,N-dimethyl, the proton peaks with chemical shifts of 6.68-7.89 ppm are the proton peaks of the aromatic ring, and the proton peak with a chemical shift of 13.11 ppm is the proton peak of indole NH. The 1H NMR spectrum is attached. Figure 1 As shown. Furthermore, its molecular weight was verified by high-resolution mass spectrometry, yielding HRMS(ESI) m / z: calcd.for C 25 H 24 N3S[M] + 398.1691, observed 398.1695, as attached. Figure 2 As shown in the figure. Analysis using 1H NMR spectroscopy and high-resolution mass spectrometry confirmed that the synthesized compound was indeed the target product, SR-In.

[0032] Example 2: Synthesis of thirodane peroxynitrite probe SR-In containing an indole base ring

[0033] 3,3'-Thiobis(N,N-dimethylaniline) (54.5 mg, 0.2 mmol), 3-indolecarboxaldehyde (87.1 mg, 0.6 mmol), and 2 mL of methanesulfonic acid were added sequentially to a thick-walled pressure-resistant tube, and the reaction was carried out at 120 °C for 5 hours. After the reaction was completed, the reaction solution was poured into 20 mL of ice water and neutralized with sodium carbonate. Dichloromethane (3 × 20 mL) was added for extraction, and the solution was dried over anhydrous sodium sulfate and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using eluent (methanol:dichloromethane = 1:20) to obtain SR-In (44.7 mg, 53%).

[0034] The characterization results of SR-In obtained in this embodiment are the same as those in Example 1, and can be referred to the appendix. Figure 1 and attached Figure 2 .

[0035] Example 3: Synthesis of thirodane peroxynitrite probe SR-In containing an indole base ring

[0036] 3,3'-Thiobis(N,N-dimethylaniline) (54.5 mg, 0.2 mmol), 3-indolecarboxaldehyde (145.2 mg, 1 mmol), and 2 mL of methanesulfonic acid were added sequentially to a thick-walled pressure-resistant tube, and the reaction was carried out at 140 °C for 8 hours. After the reaction was completed, the reaction solution was poured into 20 mL of ice water and neutralized with sodium carbonate. Dichloromethane (3 × 20 mL) was added for extraction, and the solution was dried over anhydrous sodium sulfate and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using eluent (methanol:dichloromethane = 1:20) to obtain SR-In (52.3 mg, 62%).

[0037] The characterization results of SR-In obtained in this embodiment are the same as those in Example 1, and can be referred to accordingly. Figure 1 and Figure 2 .

[0038] Example 4: Response test of thirodamine peroxynitrite probe SR-In containing an indole base ring to peroxynitrite.

[0039] The thirodamine peroxynitrite probe SR-In containing an indole base ring, prepared in Example 1, was dissolved in DMSO to prepare a 2 mM test stock solution. The test stock solution was diluted with PBS buffer at pH 7.4 to a concentration of 5 × 10⁻⁶ mM. -6 A sample solution with a concentration of mol / L was used to measure its UV-Vis absorption spectrum, fluorescence emission spectrum, and fluorescence excitation spectrum. Peroxynitrite was added to the sample solution to bring its final concentration to 5 × 10⁻⁶. -5 After reacting for 10 minutes at a concentration of mol / L, the UV-Vis absorption spectrum, fluorescence emission spectrum, and fluorescence excitation spectrum were measured.

[0040] UV-Vis absorption spectroscopy showed that the maximum absorption wavelength of SR-In was at 570 nm. With the addition of peroxynitrite, the characteristic absorption at 570 nm disappeared, and an absorption peak appeared in the 320-450 nm range, indicating that peroxynitrite can react with SR-In to form a new substance. The results are shown in [Figure number missing]. Figure 3 .

[0041] Fluorescence emission spectroscopy showed that SR-In was essentially non-fluorescent due to the action of PET, but exhibited strong fluorescence in the 450-600 nm range after reacting with peroxynitrite, with a maximum emission wavelength of 484 nm, representing a 33-fold increase in fluorescence. The results are shown in [Figure number missing]. Figure 4 .

[0042] Fluorescence excitation spectroscopy showed that the maximum excitation wavelength of SR-In after reaction with peroxynitrite was 408 nm. (See results below.) Figure 5 The maximum excitation and emission wavelengths of SR-In after reacting with peroxynitrite are consistent with those of thioxanthones, indicating that the mechanism by which SR-In detects peroxynitrite may be the oxidation of the probe to generate thioxanthones.

[0043] Example 5: Response of the thirodane peroxynitrite probe SR-In containing an indole base ring to different types of interfering substances.

[0044] The indole-containing thirodamine SR-In prepared in Example 1 was dissolved in DMSO to prepare a 2 mM test stock solution. The test stock solution was diluted with PBS buffer at pH 7.4 to a concentration of 5 × 10⁻⁶ mM. -6 Different types of interfering substances (nitrite, nitric oxide, nitrate, hypochlorite, hydroxyl radical, superoxide anion, and hydrogen peroxide) were added to the sample solution at a concentration of 5 × 10⁻⁶ mol / L to achieve a final concentration of 5 × 10⁻⁶. -5 The concentration was set to mol / L, and the excitation wavelength was set to 405 nm. The fluorescence intensity at 484 nm was measured after adding different interfering substances, and compared with the samples with added blank sample and sample with added peroxynitrite. None of the interfering substances could induce fluorescence in SR-In at 484 nm, indicating that the probe SR-In has good selectivity for peroxynitrite. The results are shown in [Figure number missing]. Figure 6 .

Claims

1. A thirodane peroxynitrite probe SR-In containing an indole base ring, characterized in that, The thiorhodamine peroxynitrite probe SR-In, which contains an indole base ring, has the following structure: 。 2. A method for preparing a thirodane peroxynitrite probe containing an indole base ring, characterized in that, The steps are as follows: 1) 3,3'-thiobis( N,N (-Dimethylaniline) and indole-3-carboxaldehyde are mixed and soluble in acidic solvents; 2) The solution obtained in step 1) is stirred and reacted under high temperature conditions, and then poured into ice water to neutralize the acidic reaction solution with sodium carbonate; in step 1), the acidic solvent is one of methanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, propionic acid, and trifluoroacetic acid. 3) After extraction with an organic solvent, the organic phase is dried and the organic solvent is removed by vacuum distillation to obtain the crude product; 4) The crude product was separated and purified by silica gel column chromatography with eluent to obtain the thiorhodamine peroxynitrite probe SR-In containing an indole base ring; The synthesis route is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step 1), 3,3'-thiobis( N,N The molar ratio of 3,3'-dimethylaniline and 3-indolecarboxaldehyde is 1:1-5, 3,3'-thiobis( N,N The concentration of dimethylaniline (DME) in acidic solvents is 20-100 mg / mL.

4. The preparation method according to claim 2, characterized in that, In step 2), the reaction temperature is 100-140℃ and the reaction time is 2-8 hours.

5. The preparation method according to claim 2, characterized in that, In step 3), the organic solvent used for extraction is one of dichloromethane, chloroform, or ethyl acetate.

6. The preparation method according to claim 2, characterized in that, The eluent is methanol and dichloromethane in a volume ratio of 1:15-30.

7. Application of a thirodamine peroxynitrite probe SR-In containing an indole base ring in the detection of peroxynitrite for non-disease diagnostic and therapeutic purposes; In the presence of peroxynitrite, the probe molecule is oxidized to thioxanthone by peroxynitrite, and the thirodamine peroxynitrite probe SR-In containing an indole base ring is excited to produce strong fluorescence in the range of 450-600 nm under 405 nm conditions, thereby realizing the detection of peroxynitrite.

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